This application is a resubmission of a proposal reviewed by the General Medicine B Study Section in Oct/Nov 1980. #1R01 AM 28420-01. 1,25(OH)2D3 is the most active form of vitamin D3 and plays a pivotal role in the maintenance of calcium homeostasis. This hormonal form of the vitamin was once thought to be synthesized solely in the kidney from 25(OH)D3. Recent evidence has now demonstrated that mammalian placenta and decidual tissue as well as chick bone and chorioallantoic membrane cells have the ability to form significant amounts of 1,25(OH)2D3 and 24,25(OH)2D3 from 25(OH)D3. This implies the presence of an active 25(OH)D3-1-hydroxylase and 25(OH)D3-24-hydroxylase in these tissues. All the above mentioned tissues are also involved in mineral homeostasis of an adult or fetal animal thorugh their ability to transmurally transport net amounts of calcium ion. My hypothesis, in this regard, is that all tissues involved in the net translocation of calcium ion contain an active l- and 24-hydroxylase. These hydroxylases function to either synthesize the important dihydroxy metabolites of vitamin d3 or alter the tissue levels of these metabolites obtained from serum. I propose to utilize new isolated cell culture techniques to explore three main aspects of the extrarenal metabolism of 25(OH)D3. First, I will carefully characterize the 1- and 24-hydroxylases reported in chick bone and chorioallantoic membrane cells in an attempt to demonstrate the presence of 1- and 24-hydroxylase activity in a mammalian bone cell system. Third, I will test for the presence of 1- and 24-hydroxylase activity in other tissues (both avian and mammalian) conforming to my hypothesis.